scholarly journals Lipidomic and Ultrastructural Characterization of Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum

2020 ◽  
Author(s):  
Kelly M. Hines ◽  
Gloria Alvarado ◽  
Xi Chen ◽  
Craig Gatto ◽  
Antje Pokorny ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Human Serum ◽  
Serum Lipids ◽  
Cell Envelope ◽  
Lipid Classes ◽  
Cholesteryl Esters ◽  
Cell Envelopes ◽  
Triton X

ABSTRACTStaphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained much fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus requires a living or non-denatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed CEs and TGs being the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host.IMPORTANCEComprehensive lipidomics of S. aureus grown in the presence of human serum suggests human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids-derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo. Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

scholarly journals Lipidomic and Ultrastructural Characterization of the Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Kelly M. Hines ◽  
Gloria Alvarado ◽  
Xi Chen ◽  
Craig Gatto ◽  
Antje Pokorny ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Human Serum ◽  
Serum Lipids ◽  
Cell Envelope ◽  
Lipid Classes ◽  
Cholesteryl Esters ◽  
Content Type ◽  
Triton X

ABSTRACT Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When S. aureus was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus require a living or nondenatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed that CEs and TGs were the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host. IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo. Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface

PROTEOMICS ◽  
2011 ◽  
Vol 11 (14) ◽  
pp. 2921-2930 ◽  
Author(s):  
Annette Dreisbach ◽  
Magdalena M. van der Kooi-Pol ◽  
Andreas Otto ◽  
Katrin Gronau ◽  
Hendrik P. J. Bonarius ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Human Serum ◽  
Serum Proteins ◽  
Versatile Tool ◽  
Human Serum Proteins

scholarly journals Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Robert C. Kuiack ◽  
Ruud A. W. Veldhuizen ◽  
Martin J. McGavin
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Antimicrobial Peptides ◽  
Sensor Kinase ◽  
Acidic Ph ◽  
Content Type ◽  
Enhanced Resistance ◽  
Secreted Proteases ◽  
Arachidonic Acids

ABSTRACT Although the GraS sensor kinase of Staphylococcus aureus is known for the sensing of and resistance to cationic antimicrobial peptides (CAMPs), we recently established that it also signals in response to acidic pH, which is encountered on human skin concurrently with CAMPs, antimicrobial unsaturated free fatty acids (uFFA), and calcium. We therefore evaluated how these environmental signals would affect GraS function and resistance to antimicrobial uFFA. Growth at pH 5.5 promoted increased resistance of S. aureus USA300 to linoleic and arachidonic acids but not palmitoleic or sapienic acid. However, enhanced resistance to these C16:1 uFFA was achieved by supplementing acidic medium with 0.5 mM calcium or subinhibitory CAMPs. Enhanced resistance to uFFA at acidic pH was dependent on GraS and GraS-dependent expression of the lysyl-phosphatidylglycerol synthase enzyme MprF, through a mechanism that did not require the lysyl-transferase function of MprF. In addition to enhanced resistance to antimicrobial uFFA, acidic pH also promoted increased production of secreted proteases in a GraS-dependent manner. During growth at pH 5.5, downstream phenotypes of signaling through GraS, including resistance to uFFA, MprF-dependent addition of positive charge to the cell surface, and increased production of secreted proteases, all occurred independently of acidic amino acids in the extracytoplasmic sensor loop of GraS that were previously found to be required for sensing of CAMPs. Cumulatively, our data indicate that signaling through GraS at acidic pH occurs through a mechanism that is distinct from that described for CAMPs, leading to increased resistance to antimicrobial uFFA and production of secreted proteases. IMPORTANCE Staphylococcus aureus asymptomatically colonizes 30% of humans but is also a leading cause of infectious morbidity and mortality. Since infections are typically initiated by the same strain associated with asymptomatic colonization of the nose or skin, it is important to understand how the microbe can endure exposure to harsh conditions that successfully restrict the growth of other bacteria, including a combination of acidic pH, antimicrobial peptides, and antimicrobial fatty acids. The significance of our research is in showing that acidic pH combined with antimicrobial peptide or environmental calcium can signal through a single membrane sensor protein to promote traits that may aid in survival, including modification of cell surface properties, increased resistance to antimicrobial fatty acids, and enhanced production of secreted proteases.

scholarly journals Plasma phospholipid fatty acids in X-linked adrenoleukodystrophy

1996 ◽  
Vol 42 (3) ◽  
pp. 454-461 ◽  
Author(s):  
M I Aveldaño ◽  
D Donnari
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Blood Cell ◽  
Blood Cells ◽  
Saturated Fatty Acids ◽  
Plasma Lipid ◽  
Plasma Phospholipid ◽  
Lipid Classes ◽  
Cholesteryl Esters ◽  
Blood Cell And Plasma

Abstract Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked adrenoleukodystrophy. The increase in saturated fatty acids with very long chains typical of this disease occurred almost exclusively in sphingomyelin. In this lipid, the proportion of lignoceric (24:0) and hexacosanoic (26:0) acids increased while that of 18:0, 20:0, and 24:1 decreased. In the rest of the lipid classes, but especially in cholesteryl esters and triacylglycerols, the proportion of linoleate (18:2) decreased while that of oleate (18:1) increased. In glycerophospholipids, polyunsaturated fatty acids such as 20:4n-6, 22:5n-6, and 22:6n-3 were reduced while their immediate precursors, 20:3n-6, 22:4n-6, and 22:5n-3, respectively, were relatively increased, suggesting a defect in fatty acid desaturation mechanisms. Although less pronounced, a similar trend of changes was seen in the patient's mother; in both, all alterations were more marked in serum than in blood cells.

scholarly journals Branched chain fatty acid synthesis drives tissue-specific innate immune response and  infection dynamics of  Staphylococcus aureus

2021 ◽  
Vol 17 (9) ◽  
pp. e1009930
Author(s):  
Xi Chen ◽  
Wei Ping Teoh ◽  
Madison R. Stock ◽  
Zachary J. Resko ◽  
Francis Alonzo
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Membrane Fluidity ◽  
Unsaturated Fatty Acids ◽  
Cell Envelope ◽  
Acyl Chain ◽  
Dependent Manner ◽  
Infection Dynamics ◽  
Branched Chain

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.

scholarly journals Assembly and mechanisms of bacterial type IV secretion machines

2012 ◽  
Vol 367 (1592) ◽  
pp. 1073-1087 ◽  
Author(s):  
Ellen L. Zechner ◽  
Silvia Lang ◽  
Joel F. Schildbach
Keyword(s):  
Cell Surface ◽  
Cell Envelope ◽  
Recipient Cell ◽  
Antibiotic Resistance Genes ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv ◽  
Wide Range ◽  
Cell Envelopes

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.

THE EFFECT ON HUMAN SERUM-LIPIDS OF A DIETARY FAT, HIGHLY UNSATURATED, BUT POOR IN ESSENTIAL FATTY ACIDS

The Lancet ◽  
1959 ◽  
Vol 273 (7064) ◽  
pp. 115-119 ◽  
Author(s):  
EdwardH. Ahrens ◽  
Jules Hirsch ◽  
William Insull ◽  
Wilhelm Stoffel ◽  
MalcolmL. Peterson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Human Serum ◽  
Dietary Fat ◽  
Serum Lipids ◽  
Essential Fatty Acids

scholarly journals Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

2006 ◽  
Vol 188 (3) ◽  
pp. 1011-1021 ◽  
Author(s):  
Valério R. F. Matias ◽  
Terry J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Envelope ◽  
High Density ◽  
Low Density ◽  
Periplasmic Space ◽  
Wall Zone ◽  
Cell Envelopes

ABSTRACT The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.

Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope

1982 ◽  
Vol 152 (2) ◽  
pp. 822-828
Author(s):  
I Moriyon ◽  
D T Berman
Keyword(s):  
Cell Envelope ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Control Cell ◽  
Sodium Dodecyl ◽  
E Coli ◽  
Alkyl Chains ◽  
Cell Envelopes ◽  
Triton X ◽  
Nonionic Detergents

Cell envelopes prepared from smooth and rough strains of Brucella were characterized on the basis of lipopolysaccharide and protein content. The action of three kinds of detergents on Brucella cell envelopes and Escherichia coli control cell envelopes was examined on the basis of the proteins and lipopolysaccharides that were extracted. As compared with those of E. coli, Brucella cell envelopes were resistant to nonionic detergents. Zwittergents 312 and 316 were most effective in extracting E. coli cell envelopes, and Zwittergent 316 was most effective in extracting Brucella cell envelopes. Sarkosyl extracted proteins but extracted only trace amounts of lipopolysaccharides from cell envelopes of both bacteria. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the Sarkosyl-resistant proteins revealed a composition similar to that of the proteins exposed on the surfaces of viable cells, as determined by the lactoperoxidase-125I radioiodination method. EDTA, with either Tris-HCl or Tris-HCl-Triton X-100, did not have detectable effects on Brucella cell envelopes. Ultracentrifugation of purified lipopolysaccharides in detergents and EDTA demonstrate that, in contrast to that of E. coli, Brucella lipopolysaccharide was not stabilized by divalent cations. Sarkosyl was ineffective in dispersing lipopolysaccharides, whereas the action of Zwittergents was related to the length of their alkyl chains.

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